3&#39;-expression enhancing fragments and method

ABSTRACT

The invention concerns a method for extending the half-life of mRNAs. The half-life extension is conferred upon the mRNA by a co-transcribed positive retroregulatory element which is ligated to the 3&#39; end of the DNA sequence encoding the RNA. RNAs having an extended half-life conferred by a co-transcribed positive retroregulatory element are also claimed.

This application is a continuation-in-part application of copending U.S.Ser. No. 646,584, filed Aug. 31, 1984, U.S. Pat. No. 4,792,523.

FIELD OF THE INVENTION

The invention concerns the field of recombinant DNA. More particularlythe invention concerns positive retroregulatory elements, which whenligated to selected DNA sequences coding for a gene product, enhance theexpression of the gene product. Plasmids carrying the positiveretroregulatory element ligated to selected DNA sequence and cellstransformed by such plasmids are provided. In addition, the inventionrelates to a method for enhancing expression of a gene product byligating a positive retroregulatory element to a selected DNA sequenceexpressionable for a desired gene product.

One of the fundamental strategies forming the basis for the commercialutility of recombinant DNA technology is the production in relativelyhigh volumes of gene products in the form of polypeptides or proteinsthat ordinarily occur in nature at very low concentrations or volume. Bytransforming cells that have relatively short generation or doublingtimes with recombinant molecules, which generally are in the form ofplasmids, significant amounts of a desired gene product may be produced.Although each cell harboring the recombinant DNA molecule may in factproduce only a very small amount of the desired gene product, the rapidmultiplication of the cell allows the production of significant amountsof the desired gene product.

In general, the goal of producing larger amounts of a selected geneproduct is limited by the size of culture medium volumes required forgrowing the transformed cell that produces the desired gene product.Increasing the yield of a desired gene product is clearly a majorconcern in the commercial production of a desired gene product. Oneapproach to increasing yield is to improve the recovery rate of thedesired gene product from the transformed cell or culture medium whileleaving the amount of product produced by the cell unchanged. Thisapproach entails the addition of processing steps to increase recoveryof the desired gene product from a given fermentation run. Suchadditional processing steps can require enormous costs for addedequipment and personnel.

A different approach to increasing yield of a desired gene product is toincrease the expression of the desired gene product by the transformedcell. Such increases in production of the desired gene product by eachcell can lead to increases in yield for a particular fermentation run aswell as improvements in purity of the product produced and a lowering ofthe cost per unit of product since further processing may beunnecessary.

One of the important general approaches to improving the yield ofdesired gene product per cell is to provide optimal culture conditionsfor the cell. By enriching the nutrient content of the medium, providingoptimal temperatures for fermentations, furnishing the optimum amount oftrace factors and supplementing the culture medium with required aminoacids, for example, yields can be significantly improved.

A second general approach to the problem of increasing the yield of adesired gene product per cell is to manipulate the regulatory elementscontrolling the expression of the gene product by the cell. One methodof manipulating the regulatory elements controlling expression of adesired gene product is by the selection of strong promoters. Promotersmay be generally defined as regions of a DNA molecule to which RNApolymerase binds to initiate transcription of messenger RNA (mRNA) froma DNA sequence coding for a gene product. Strong promoters have thecharacteristic of initiating an RNA transcript by RNA polymerase withhigher frequency than weaker promotors with the result that the DNAsequence with which the promoter is associated is transcribed to formmore mRNA transcript coding for the desired gene product.

It has become conventional in the recombinant DNA field to ligate astrong promoter to a selected DNA sequence coding for a desired geneproduct, in proper reading frame such that mRNA transcripts, initiatedfrom the strong promoter are produced. Multiple strong promoters may beligated to a selected DNA sequence, thereby increasing the opportunityfor binding RNA polymerase to the DNA sequence and producing more mRNAtranscript from the DNA sequence. An example of this last method is theuse of tandem lac operon promoters at the beginning of a DNA sequencecoding for a desired gene product.

Another approach for increasing the binding of RNA polymerase to providehigher levels of mRNA transcript is the elimination of regulatoryfactors that tend to reduce the ability of RNA polymerase to transcribemRNA from a DNA sequence coding for a desired gene product. Certainstrong promoters have associated with them attenuator regions whichunder certain conditions cause a bound RNA polymerase to ceasetranscription of DNA sequences with which the attenuator regions areassociated. One such attenuator region is associated with the promoterof the tryptophan operon, a promoter which is known to be a strongpromoter. By eliminating the attenuator region of the promoter of thetryptophan operon, the tryptophan promoter can serve as an unimpairedstrong promoter.

Another approach to increasing the yield of a desired gene product is toligate strong ribosome binding sites within a selected DNA sequence suchthat ribosomes bind with high efficiency to the mRNA transcript that hasbeen transcribed from the selected DNA sequence. By increasing theaffinity of an mRNA transcript for the ribosome through such strongribosome binding sites, it is believed translation of the mRNAtranscript occurs with a greater frequency, thus increasing theproduction of the desired gene product.

All of the above-mentioned techniques for increasing the expression of adesired gene product involve manipulation of regulatory sequences thatappear at the 5' end of the DNA sequence coding for the desired geneproduct. In all cases, these regulatory sequences are involved in eitherinitiation of transcription of the DNA sequence coding for the desiredgene product, or initiation of translation of the mRNA transcriptcorresponding to the DNA sequence coding for the desired gene product.

Another form of regulation of the expression of a gene has been observedin various viral systems. The term "retroregulation" has been coined forthis form of expression control. Court et al. infra (1983). As describedin the prior art, the known form of retroregulation is uniformly foundto decrease the expression of the gene product on which theretroregulator exerts its effect. Thus, known forms of retroregulationare negative retroregulation.

In bacteriophage λ, the regulation of the expression of the integrasegene (int) mRNA sequence by a 3'-terminal sequence designated sib, isdescribed in Schindler and Echols, "Retroregulation of the int gene ofbacteriaphage λ: Control of translation completion", Proc. Natl. Acad.of Sci. (USA), 78:4475-4479 (1981). Schindler and Echols postulate thatthe sib region acts at the translation level of expression by preventingnormal completion of protein synthesis from int mRNA. It is furtherpostulated that the sib regulatory region corresponds to an mRNA regionwhich is able to form a stem and loop secondary structure duplex. Thesib region also is believed to include a region near the end of the genewhich it regulates that provides a cleavage site for RNase III. Thiscleavage site for RNase III renders the mRNA sensitive to degradation byvarious exonucleases. The authors postulate that by degrading the mRNAtranscript, protein synthesis of the int gene product is prematurelyterminated. Thus, the reference discloses a negative regulatory role forthe sib regulatory sequence, whereby the production of the polypeptideencoded by the int gene is decreased. Guarneros et al.,Posttranscriptional Control of Bacteriophage λ int Gene Expression Froma Site Distal to the Gene, Proc. Natl. Acad. of Sci. (USA), 79:238-242(1982) discloses that in bacterial hosts which lack functional RNaseIII, sib regulation is defective. The reference further concurs withSchindler and Echols, supra, that the sib retroregulatory elementfunctions post-transcriptionally to prevent mRNA translation byincreasing the degradation of int mRNA. Court et al., Detection Analysisof the Retroregulatory Site for the λ int Gene, J. Mol. Biol.,166:233-240 (1983) concurs with the general teachings of Guarneros etal. supra and further suggests that the RNase III sensitive structure ofthe retroregulatory element encoding the sib site is similar to otherRNase III sensitive sites found in Escherichia coli and phage.

In a general discussion of termination of transcription in E. coli,Holmes et al., Termination of Transcription in E. coli, Cell,32:1029-1032 (1983), discloses that the primary structure of DNAsequences coding for transcription terminators includes runs of adenineand thymine base pairs on either side of symmetrical guaninecytosinerich sequences. mRNA transcripts from these sequences form secondaryhairpin loop or stem and loop structures which are followed by runs ofuridine residues on either side. Such terminators function asterminators in either orientation. The reference, however, emphasizesthat the significance of this bidirectional terminator activity isunclear. The reference also discusses the fact that RNA transcripts ofDNA sequences coding for transcription terminators that lack a stem andloop structure, are degraded more rapidly than terminators having suchstructures. Platt and Bear in "Role of RNA Polymerase, p Factor andRibosomes in Transcription Termination", Gene Function in Prokaryots,Beckwith, et al. eds., Cold Spring Harbor Laboratory, N.Y. (1983)generally reviews transcription termination and the role of secondarystructures at the end of RNA transcripts in the regulation oftranscription termination. The authors speculate on the possibility ofseveral functions for secondary structures in mRNA, one of whichincludes the stabilization from degradation beyond a certain point by 3'exonucleases of completed mRNA transcripts. There is, however, nosuggestion that enhancement of expression of the gene product for whichthe mRNA transcript codes is obtained.

Gentz, R., Cloning and Analysis of strong promoters is made possible bythe downstream placement of a RNA termination signal, Proc. Natl. Acad.of Sci. (USA), 78:4936-4940 (1981) shows that in plasmid pLBU3, additionof a strong transcription terminator derived from the bacteriophage fd,to the alpha fragment of the β-galactosidase gene which lacked the lacpromoter region, made possible the cloning of strong promoters fromphage T5. Strong promoters were isolated by increased β-galactosidaseactivity in an M15 (a lac α fragment deletion mutant) complementationassay. Strong transcription termination activity was shown by the fdterminator, which has a region of dyad symmetry, but this strongtranscription termination activity was only reported in an orientationopposite to its native orientation in the fd genome in the systemdescribed by Gentz et al. The fd fragment in pLBU3, unlike its activityin the fd genome, cooperates with p factor to terminate transcriptionefficiently. There is however no teaching, nor is any evidence shown byGentz et al., that the fd transcription terminator itself has anenhancing effect on expression of the β-galactosidase gene. Gentz et al.attribute the assayable β-galactosidase activity to the strong promotersderived from phage T5 which are ligated to 5' end of the DNA sequencecoding for the α fragment of the β-galactosidase gene.

Flock et al., Expression in Bacillus subtilis of the gene for humanurogastrone using synthetic ribosome binding sites, Molecular andGeneral Genetics, 195:246-251 (1984) state that "placing a transcriptionterminator from bacteriaphage fd immediately after the urogasterone genein (plasmid) pFF810 improves the overall expression about 5-10 times inE. coli." No details of which fd transcription terminator signal, itssequence, orientation or placement relative to immediate end of theurogasterone gene are given. The fd transcription terminator of Flock etal. is used in conjunction with a putatively strong promoter which isexpected to be highly active in B. subtilis.

In some bacteria relatively stable high copy number mRNAs have beenobserved that appear to be associated with bacterial proteins orlipoproteins that occur in large amounts. For example, it has beenreported that mRNA from the cry gene of Bacillus thuringiensis has alonger half-life than other mRNAs of genes expressed during sporulation.Petit-Galtron, M. F. and Rapoport, G., Translation of a stable mRNAfraction from sporulating cells of B. thuringiensis in a cell-freesystem from E. coli, Biochimie, 58:119-129 (1976). This increased mRNAstability has long been speculated as the contributing factor for themassive synthesis of the crystal protein during sporulation.

In addition, the mRNA from the lipoprotein (lpp) gene of E. coli whichdirects the synthesis of a major outer membrane protein is known to berelatively stable and has been shown to have sequences capable offorming extensive secondary structures in the form of stem and loopstructures having ΔG° values ranging from about -0.4 to about -21.1kcal/mole. Nakamura, K. et al., Messenger ribonucleic acid of thelipoprotein of the E. coli outer membrane II. The complete nucleotidesequence, J. Biol. Chem., 255:210-216 (1980). Neither Nakumura et al.nor Petit-Galtron et al. suggest that these structures may be used toenhance expression of a desired gene product. Moreover, the nucleotidesequence for the cloned cry gene, as will be shown hereinbelow, lacksthe extensive secondary structure associated with the mRNA of the lppgene.

In summary, the prior art with respect to retroregulation in general,shows that known retroregulatory sequences have a negative effect on theexpression of the gene which they retroregulate. With respect toterminators, Gentz et al. and Flock et al. indicate that in associationwith a DNA sequence under the control of a strong promoter, effectiveexpression of the gene for which the DNA sequence codes may be obtained,but that in the absence of such strong terminators the activity of aputative strong promoter cannot be clearly demonstrated.

DESCRIPTION OF THE INVENTION

The inventors have discovered positive retroregulatory elements which,when ligated to a DNA sequence coding for a selected gene product,significantly increase the production of the selected gene product.

As used herein, the term "selected" or "desired gene product" is meantto denote a polypeptide or protein produced by a prokaryotic oreukaryotic host by virtue of its transformation with a recombinant DNAmolecule comprising a DNA sequence coding for the polypeptide orprotein. Such a selected or desired gene product may be one otherwiseordinarily produced by the host, by a prokaryotic organism other thanthe host, or by a eukaryotic organism other than the host. The term"gene" as used herein means a DNA sequence coding for a polypeptide orprotein. The term "expression" as used hereinbelow refers to theproduction of a polypeptide or protein coded for by a DNA sequence orgene. In general, the positive retroregulatory element is ligateddownstream of the DNA sequence coding for the selected gene product. Asused herein, the term "downstream" is used with respect to the directionof transcription of the messenger RNA to which the DNA sequencecorresponds, transcription proceeds from upstream to downstream.

The location of the positive retroregulatory element is generally 3' tothe end of the coding strand of the DNA sequence coding for the selectedgene product. It is well known that in order for a gene to be expressed,a translation termination codon is usually found at the 3' end of theDNA sequence coding for the selected gene product. Typically, thepositive retroregulatory element is ligated to a DNA sequence coding fora selected gene product 3' to the translation termination sequenceassociated with the DNA sequence for the selected gene. Knowntranslation termination sequences generally are nucleotide triplets. Anexample of such translation termination codons include those having thesequence TAG, TAA, and TGA, wherein the letters correspond to thymine,adenine and guanine residues which are components of the DNA molecule.Translation termination codons may appear singly, paired sequentially,or in pairs having a number of nucleotides in between. See for exampleWatson, J. D., Molecular Biology of the Gene, W. A. Benjamin, Inc.,Menlo Park, Calif. (3rd ed. 1977). The translation termination signalmay be native to the DNA sequence coding for the selected gene productor may itself be ligated at the 3' end of the DNA sequence coding forthe selected gene product to provide a translation termination signal ina desired location.

The distance of the positive retroregulatory element from the 3' end ofthe gene to which it is ligated may be varied while still exerting apositive or enhancing effect on the expression of the gene. As will beexplained in greater detail herein below, a particular region of thepositive retroregulatory element isolated from the 3' flanking region ofthe gene coding for the B. thuringiensis crystal protein, which isbelieved to form a "stem and loop" structure at the end of the mRNAtranscript for the selected gene, has been ligated from about 30 toabout 300 nucleotides from the 3' end of the selected gene. In bothconstructions, a positive retroregulatory effect is shown on theexpression of the gene to which the positive retroregulatory element wasligated.

As will be shown in detail in the examples hereinbelow, positiveretroregulatory elements have been shown to enhance expression of thegene to which they are ligated irrespective of the orientation of thepositive retroregulatory element. It has been shown that so long as thepositive retroregulatory element is ligated at the 3' end of a selectedgene, the positive retroregulatory element may have a 3'-5' orientationor a 5'-3' orientation and still exert an enhancing effect on expressionof the selected gene.

The positive retroregulatory element according to the invention may becharacterized structurally as a DNA sequence which transcribes for acorresponding RNA transcript which is capable of forming a stem and loopstructure having a Gibbs free energy constant (ΔG°) of about -30.4 Kcalas determined by the methods of Tinoco, I. et al., Nature New Biology,246:40-41 (1973). In one effective embodiment, the positiveretroregulatory element according to the invention is a DNA sequencethat transcribes for a corresponding RNA transcript that is capable offorming a stem and loop structure which has a guanine-cytosine residuecontent of about 43%. It is expected that such stem and loop structureshaving guanine-cytosine contents of from about 40% to about 45% willalso be effective positive retroregulatory elements. Such positiveretroregulatory elements are intended to be within the scope of theinvention.

In a preferred embodiment of the invention, the positive retroregulatoryelement is characterized as a DNA sequence having an inverted repeatsequence including the deoxyribonucleotide sequenceAAAACGGACATCACCTCCATTGAAACGGAGTGATGTCCGTTTT wherein the underlinedportion of the sequence comprises the inverted repeat sequence. Thecorresponding mRNA transcript for the above-mentioned inverted repeatsequence has the ribonucleotide sequence AAAACGGAAUCACCUCCAUUGAAACGGAGUGAUGUCCGUUUU wherein the underlined portion of the ribonucleotidesequence is the inverted repeat sequence.

It is expected that some variants of the above-mentioned deoxynucleotidesequence and the corresponding RNA transcript which can arise eitherthrough random mutation, point mutation, addition mutation, deletionmutation, synthetic oligonucleotide directed mutagenesis or constructionof synthetic oligonucleotides may have inverted repeated sequences thatwill preserve the positive retroregulatory effect of the positiveretroregulatory element. Such variants that preserve the positiveretroregulatory effect of the positive retroregulatory element areclearly within the scope of the invention.

As will be described in greater detail hereinbelow, the positiveretroregulatory element according to the invention may be isolated froma portion of the 3' flanking region of the gene coding for the B.thuringiensis crystal protein (cry gene). The 3' flanking region may bepurified using conventional gel electrophoresis techniques and clonedinto an appropriate host for multiplication. In one embodiment of theinvention, as further described hereinbelow in the examples, a portionof the 3' flanking sequence of the cry gene which includes thetranscription terminator signal thereof, was isolated, purified andcloned. This portion of the cry gene, about 382 nucleotides in length,includes a positive retroregulatory element as previously describedhereinabove and portions of the 3' flanking region of the cry gene thushave a positive retroregulatory effect.

When the positive retroregulatory element is ligated to a DNA sequenceexpressionable for a selected gene product in the proper relationshipthereto, as described hereinabove, expression of the selected geneproduct is enhanced. Thus, the invention includes a method of enhancingexpression of a selected gene product. As used herein the term "DNAsequence expressionable for a selected gene product" is intended to meana selected gene, for example betalactamase or interleukin-2, havingassociated therewith a promoter, transcription start signal, ribosomebinding site, translation start signal and translation terminationsignal all the proper relationship and reading frame such that theproduct for which the DNA sequence codes may be expressed. Numerousappropriate promoters are well known to those skilled in the art towhich the invention pertains, and include: promoters derived from E.coli including the tryptophan promoter, lac promoter-operator andnumerous other promoters well known to those skilled in the art;Bacillus promoters including the penP promoter; and various viralpromoters such as for example the SP82 promoter and PL promoter of phageλ. Viral promoters may be recognized by various cells depending upon theparticular cell it ordinarily infects. The selected DNA sequence mayfurther include a signal sequence, for example, the penP signal sequencesuch that the expressed gene product is secreted. If the selected DNAincluding a signal sequence is expressed in E. coli, the gene productwill generally be secreted into the periplasmic space of the cell, andcan be released from the periplasmic space by sonication. If theselected DNA including a signal sequence is expressed by B. subtilis forexample, the selected gene product will generally be secreted into theculture medium and may be recovered therefrom.

The term "DNA sequence expressionable for a selected gene product" isfurthermore intended to include factors required for expression of thegene product for which the DNA sequence codes, such as enzymes includingfunctional RNA polymerase, transfer RNAs, amino acids, ribosomes andother elements necessary for the transcription and translation of theselected DNA sequence.

From the foregoing it will be understood that a "DNA sequenceexpressionable for a selected gene product" includes a vector such as aplasmid and a host cell transformed thereby which is capable ofexpressing the gene and forming the gene product coded by the gene.Examples of such plasmids and hosts are well known to those skilled inthe art and are exemplified in detail in the examples hereinbelow. Amongappropriate hosts for plasmids carrying a DNA sequence expressionablefor a selected gene product are prokaryotic and eukaryotic cells.Prokaryotes may be defined as organisms that lack a true nucleus, thenuclear membrane being absent and the nuclear structures being collectedin a nuclear region or nucleoid. The chromosomes of prokaryotes aregenerally not associated with proteins.

Among appropriate prokaryotic cells as hosts for plasmids are bothGram-positive and Gram-negative bacteria. By the terms Gram-positive andGram-negative is meant cells capable of taking up and retaining Gramstain and cells incapable of retaining Gram stain respectively. Amongappropriate Gram-positive bacteria are those belonging to the genusBacillus and in particular B. subtilis. B. subtilis strain PSL1 (BGSCnumber IA510) are particularly preferred.

Among appropriate Gram-negative bacteria are those belonging to thegenus Escherichia, and in particular E. coli strain MM294 and CS412.

Eukaryotic cells may be defined as cells having a true nucleus bound bya nuclear membrane within which are found chromosomes usually bound toproteins. Included in eukaryotic cells are plant animal and fungalcells. Among the fungal cells are yeasts, and in particularSaccharomyces and especially Saccharomyces cerevisiae are useful in thepractice of the invention.

Appropriate plasmids for carrying DNA sequences expressionable for aselected gene product are those capable of transforming a host cell suchthat the DNA sequence is expressed thereby. In general, a DNA sequenceexpressionable for a selected gene product ligated to a positiveretroregulatory element may be placed in any plasmid capable ofexpressing the gene product in an appropriate transformed host. Thus theinvention includes plasmids carrying DNA sequences expressionable for aselected gene product ligated to a positive retroregulatory element aswell as the host cell transformed therewith.

Plasmids capable of transforming E. coli include for example Col E1 typeplasmids in general. Other appropriate plasmids for transforming E. coliinclude: pSC101, pSF2124, pMB8, pMB9, pACYC184, pACYC177, pCK1, R6K,pBR312, pBR313, pBR317, pBR318, pBR320, pBR321, pBR322, pBR333, pBR341,pBR345, pBR350, pBR351, pML2, pML21, ColE1AP, RSF1010, pVH51, pVH151,and pVH153. See, Recombinant Molecules Impact on Science and Society,Beers, R. F. and Bassett, E. G., eds. Raven Press, N.Y. (1977). Theplasmids of the type pBR include pBR325, pBR327 and pBR328. See, Soberonet al., Gene, 9:287-305 (1980). Other appropriate plasmids are describedin DNA Insertion Elements, Plasmids and Episomes, Bukhari et al. (eds)Cold Spring Harbor Laboratory (1976).

Plasmids capable of transforming B. subtilis include: pC194, pC221,pC223, pUB112, pT127, pE194, pUB110, pSA0501, pSA2100, pTP4, pTP5 (seeGryczan, T. J., "Molecular Cloning in B. subtilis in The MolecularBiology of the Bacilli, Dubnau, D., Ed., Academic Press, Inc., N.Y.,1982, p. 310) and their derivatives.

Plasmids capable of transforming both B. subtilis and E. coli that maybe used in the practice of the invention include: pDH5060, pLP1201(Ostroff et al., infra, p062165 (Gray, O. and Chang, S., "Molecularcloning and expression of B. licheniformis β-lactamase gene in E. coliand B. subtilis", J. Bacteriol., 145:422-428 (1982), pHV11, pHV12,pHV14, pHV16 (Ehrlich, S. D., "DNA cloning in B. subtilis", Proc. Natl.Acad. Sci. USA, 75:1433-1436 (1978), and pSYC310-2 (McLaughlin et al.,infra. See also Old, R. W. and Primrose, S. B., "Plasmid vectors forcloning in microbes other than E. coli", Principles of Gene Manipulation2nd ed., Carr, N.6, Ingraham, L. L., and Rittenberg, S. C., Eds,University of Ca. Press, Berkeley, 1981, p. 48).

Plasmids capable of transforming S. cerevisiae include: pMP78, YEp13,pBTI1, pLC544, YEp2, YRp17, pRB8 (YIp30), pBTI7, pBTI9, pBTI10, pAC1,pSLe1, pJDB219, pDB248 and YRp7.

The DNA sequence coding for an expressionable gene product which isligated to the positive retroregulatory element may be cistronic, i.e.,coding for a single polypeptide or polycistronic, i.e., coding for aplurality of polypeptides, the mRNA for the polycistronic DNA beingunder the transcriptional control of a single promoter. Suchpolycystronic DNA sequences are well known to those skilled in the artand include, for example, the genes for tryptophan biosynthesis underthe control of the tryptophan operon in E. coli. Polycistronic genes mayalso be artificially formed by ligating a series of desired genestogether under the control of a single promoter. See for example Flock,J. et al., supra (1984).

The selected DNA sequence expressionable for a desired gene productligated to a positive retroregulatory element may be homologous to theDNA of the host or alternatively heterologous to the DNA of the host.Thus, for example, the selected DNA sequence may be derived from anorganism that is of the same species as the host cell that has beentransformed to express the gene product, in which case, the selected DNAsequence as used herein is termed homologous. An example of anhomologous gene sequence expressionable for a selected gene productaccording to the invention is the enhanced expression in an E. coli hosttransformed by a plasmid bearing a gene native to E. coli. An example isthe production of E. coli β-galactosidase wherein the enhancedexpression is mediated by the ligation of a positive retroregulatoryelement at the 3' end of the β-galactosidase.

As mentioned above, the selected DNA sequence expressionable for adesired polypeptide ligated to a positive retroregulatory element mayalternatively be heterologous to the host. Numerous examples of suchheterologous enhanced expression are given in detail hereinbelow andinclude: the enhanced expression, under mediation of a positiveretroregulatory element, of the B. licheniformis penicillinase gene inB. subtilis; eukaryotic genes such as mammalian interleukin-2 andmutated interleukin-2 in prokaryotic microorganisms such as B. subtilisand E. coli.

In another aspect of the invention the positive retroregulatory elementmay be used in a method to extend the half-life of the mRNA transcriptencoded by a selected DNA sequence. As is shown in detail in theexamples, when the positive retroregulatory element is ligated at the 3'end of a selected DNA sequence, the half-life of the mRNA transcriptencoded by the selected DNA sequence is extended. The mechanism by whichthe positive retroregulatory element exerts this mRNA half-lifeextension, is not entirely understood. One possible explanation for theobserved mRNA half-life extension is that the RNA transcript of thepositive retroregulatory element assumes a secondary structure thatprevents enzymatic hydrolysis of the mRNA transcript initiated at the 3'end of the transcript. The method of extending the half-life of mRNAtranscripts has been demonstrated using the positive retroregulatoryelement in both its 3' to 5' and 5' to 3' orientations.

The positive retroregulatory element may be used to the extend thehalf-life of the mRNA transcript encoded by a selected DNA sequence whenthe DNA sequence is heterologous to the host organism. Furthermore, thehalf-life extension of the mRNA transcript provided by the use of thepositive retroregulatory element, occurs even where the promotor,selected DNA sequence and positive retroregulator are all heterologousto the host organism. Contemplated within the scope of the invention aremethods for mRNA half-life extension in which the promotor, selectedDNA, or positive retroregulatory element are each either singly or takentogether not heterologous to the host organism.

mRNA transcripts encoded by a selected DNA sequence having the positiveretroregulatory element at the 3' end thereof, have been demonstrated tohave extended half-lives in microorganisms in which they have beentranscribed. Such microorganisms thus provide a means for transcribing aselected DNA into RNA. Other means for transcribing a selected DNA intoRNA are contemplated to be useful within the scope of the invention.Thus, eukaryotic cells such as Saccharomyces or Xenopus oocytes areconsidered as such means for transcribing DNA. In addition, cell freesystems having therein the components necessary for RNA transcriptionsuch as buffers, DNA dependent RNA polymerase, ions and ribonucleotidetriphosphates are contemplated to be useful for transcribing a selectedDNA sequence having a positive retroregulatory element ligated at the 3'end thereof to form an RNA having an extended half-life.

The extension of the half-life of the mRNA transcribed from a selectedDNA sequence having the positive retroregulatory element at the 3' endthereof, may be measured directly by hybridization of radiolabeled DNAprobes to the mRNA of cells which have been treated with transcriptioninhibitors such as rifampicin. It has been shown that enchancedexpression of a selected DNA sequence obtained by use of the positiveretroregulatory element correlates substantially with the extension ofthe half-life of the mRNA encoded by the selected DNA sequence.

From the foregoing summary of the invention, it will be clear to thoseordinarily skilled in the art that the inventors have provided positiveretroregulatory elements, plasmids carrying the positive retroregulatoryelements ligated to a DNA sequence expressionable for a selected geneproduct such that expression of the selected gene product is enhanced,cells which transformed by such plasmids express the selected geneproduct at enhanced levels, and the selected gene products so expressed.

It will furthermore be clear to those skilled in the art that theinventors have provided a general method for enhancing the expression ofa selected gene product, the method comprising providing a DNA sequenceexpressionable for a selected gene product and ligating a positiveretroregulatory element to the DNA sequence in a relationship theretowhereby expression of the selected gene product is enhanced. The generalmethod, described hereinabove and in greater detail hereinbelow in theexamples that follow, is effective to enhance expression of DNAsequences coding for a desired gene product in prokaryotic andeukaryotic hosts, and appears to be effective whether the DNA sequenceto be expressed is homologous or heterologous to the host cell thatexpresses it. The invention also includes the gene products produced bythe general method for enhancing expression of a selected gene product.

The following examples are intended by the inventors to be merelyexemplary of the invention and are not intended by the inventors to belimiting. As mentioned above, the invention described herein and claimedbelow is broadly applicable to the enhanced expression of numerous geneproducts by numerous cell types, a fact which will be readily apparentto the ordinarily skilled practitioner. The examples hereinbelow aremerely intended to provide a detailed and practical description of theinvention as applied to the cells and plasmids exemplified below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the restriction map of therecombinant plasmid pES1 bearing the cloned cry gene from B.thuringiensis. Transcription of the cry gene originates at the siteabout 350 bp upstream of the fourth EcoRI site and terminates about 360bp downstream of the third PvuII site shown in FIG. 1

The nucleotide sequence of the PvuII-NdeI restriction fragment carryingthe transcriptional terminator of cry is also shown in FIG. 1. There isa inverted repeat sequence located approximately 40-bp upstream of theNdeI site. An mRNA transcript made from this region can potentially forma stem and loop structure.

FIG. 2 shows the expected stem and loop structure of the RNA coded bythe inverted repeat sequences indicated in FIG. 1. The stem-and-loopstructure is relatively rich in G/C, about 44% G/C, with a predicted ΔG°of about -30.4 Kcal as calculated by the rules of Tinoco, et al., supra.

FIG. 3 is a schematic representation of plasmid pSYC667.

FIG. 4 is a schematic representation of plasmids pLW1, pHCW701 andpHCW702.

FIG. 5 is a schematic representation of plasmids pHCW300 and pHCW301 andthe plasmids from which they were constructed.

FIG. 6 is a schematic representation of plasmid pFC54.t. None of theplasmids shown in the figure are intended to be scale drawings. Relativepositions of endonuclease positions and relevant coding regions of theplasmids are depicted.

FIG. 7 is a graph of the decay of the penP mRNAs produced by E. coli andB. subtilis harboring the plasmids pSYC667 and pHCW-A3.

The upper curves of FIG. 7A and 7B, having t1/2 s of six minutes are thepenP mRNA decay curves in E. coli and B. subtilis of pHCW-A3 which hasthe positive retroregulator. The lower curves of FIGS. 7A and 7B havingt1/2 s of about two minutes are the penP mRNA decay curves in E. coliand B. subtilis pSYC667 which lacks the positive retroregulator.

The following is a list of restriction endonucleases that were used inthe examples appearing hereinbelow. The left column of the list is thecommonly used name of the particular endonucleases and the right columnis the organism from which it was originally derived. The listedrestriction endonucleases were used under the buffer and temperatureconditions recommended by the supplier. Restriction endonucleases areavailable from numerous commercial vendors including New EnglandBioLabs, Beverly, Mass., USA and Bethesda Research Laboratories,Gaithersberg, Md. USA. The identity of the nucleotide sequences at whichparticular restriction endonucleases cut may be found in productcatalogues of suppliers of the enzymes, as well as standard referencetexts.

    ______________________________________                                        Restriction Enconuclease                                                                      Organism Source                                               ______________________________________                                         ##STR1##       Anabaena variabilis                                            ##STR2##       Arthobacter luteus                                             ##STR3##       Bacillus amyloliquefaciens                                     ##STR4##       Bacillus aneurinolyticus                                       ##STR5##       Bacillus caldolyticus                                          ##STR6##       Caryophanon latum                                              ##STR7##       Escherichia coli                                               ##STR8##       Haemophilus haemolyticus                                       ##STR9##       Haemophilus haemolyticus                                       ##STR10##      Haemophilus haemolyticus                                       ##STR11##      Neisseria denitrificans                                        ##STR12##      Nocardia rubra                                                 ##STR13##      Escherichia coli                                               ##STR14##      Proteus vulgaris                                               ##STR15##      Staphylococcus aureus                                          ##STR16##      Serratia marcescens                                            ##STR17##      Streptomyces phaeochromogenes                                  ##STR18##      Steptomyces tubercidicus                                       ##STR19##      Xanthomonas badrii                                            ______________________________________                                    

Certain phrases and abbreviations are used herein. Unless otherwisenoted, the meaning intended are as follows:

As used herein, the term "penP" is meant to denote the prepenicillinasegene of B. licheniformis strain 749/C, or, where clear from the context,a relevant portion thereof. The nucleotide sequence of penP has beenpublished by Kroyer, J., and Chang, S., Gene, 15:343-347 (1981), andNeugebauer, K., Sprengel, R., and Schaller, H., Nucl. Acids Res.,9:2577-2589 (1981).

As used herein, "codon" means, interchangeably, (i) a triplet ofribonucleotides in an mRNA which is translated into an amino acid in apolypeptide or a code for initiation or termination of translation, or(ii) a triplet of deoxyribonucleotides in a gene whose complementarytriplet is transcribed into a triplet of ribonucleotides in an mRNAwhich, in turn, is translated into an amino acid in a polypeptide or acode for initiation or termination of translation. Thus, for example,5'-TCC-3' and 5'-UUC-3' are both "codons" for serine, as the term"codon" is used herein.

As used herein, "nucleotide", "deoxynucleotide", and"deoxyribonucleotide" all mean deoxyribonucleotide.

dNTP or NTP means any of the deoxyribonucleotide triphosphates, i.e.,ATP, GTP, CTP or TTP.

"bp" means base pair, and "kb" means kilobase pairs.

"Polypeptide" means any peptide with two or more amino acids, includingproteins.

"Coding sequence" or "DNA coding sequence" means a DNA sequence encodinga polypeptide.

"ATCC" means American Type Culture Collection, Rockville, Md. USA. Whenused in connection with a number, for example "ATCC 37017", ATCC refersto the American Type Culture Collection accession number for an organismon deposit with the ATCC.

"Operably linked" when used in regard to DNA sequence refers to thesituation wherein the sequences are juxtaposed in such a manner so as topermit their ordinary functionality. For example, a promoter operablylinked to a coding sequence refers to those linkages where the promoteris capable of controlling the expression of the sequence. The promotersoperably linked to a ribosome binding coding sequence has the samesignificance: i.e., it permits the ribosome binding site (RBS) to bepositioned in the transcript so as to participate in the initiation ofthe translation of the RNA transcript. An RBS operably linked to a startcodon is positioned so as to permit the start of translation at thiscodon.

The methods of the present invention make use of techniques or geneticengineering and molecular cloning. General techniques of geneticengineering and molecular cloning are included in Maniatis, T., Fritsch,E. F., and Sambrook, J., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, 1982, and Methods in Enzymology, Volume 68,Recombinant DNA, (Wu, R., editor), Academic Press, N.Y., 1979.

Oligonucleotide synthesis may be carried out by a number of methodsincluding those disclosed in U.S. Pat. No. 4,415,734, and in Matteuci etal., J. Am. Chem. Soc., 103 (11):3185-3191 (1981), Adams et al., J. Am.Chem. Soc., 105 (3):661-663 (1983, and Bemcage et al., TetrahedronLetters, 22 (20):1859-1867 (1981).

EXAMPLE I Preparation of 3'-Expression Enhancement Fragments

A. pES1

Plasmid pES1 was prepared as described in published European PatentApplication Publication 82302137.3, which is herein incorporated byreference, from plasmid pBR322 (ATCC 37017) and a digest of the largeplasmid fragment of B. thuringiensis var. Kurstaki HD-1. pESI is alsodescribed in H. Schnepf and H. Whiteley, Proc. Natl. Acad. Sci. (USA),78:2893-2897 (1981), and U.S. Pat. No. 4,448,885 which is hereinincorporated by reference. pES1 in E. coli K-12/HB101 has been depositedby the assignee of the above-cited European application at the AmericanType Culture Collection, Rockville, Md. under the terms of the BudapestTreaty. The deposit number is ATCC 31995. B. thuringiensis var. Kurstakistrain HD-1-Dipel is available from the Northern Regional ResearchLaboratory Culture Collection in Peoria, Ill. under no. B3792. B.thuringiensis var. Kurstaki HD-1 is available from the American TypeCulture Collection under accession number 1615.

As described in Schnepf and Whiteley, supra, and in H. Wong et al.,Jour. Biol. Chem., 258, 1960 (1983), pES1 has inserted into the BamHIsite on its pBR322 parent, a Sau3A1 fragment from the 30-megadaltonplasmid, the 47-megadalton plasmid, or both such plasmids from B.thuringiensis. The Sau3A1 fragment includes a gene coding for thedelta-endotoxin crystal protein of B. thuringiensis (cry gene). Thisgene is expressed to form a biologically active crystal protein in E.coli transformed with the plasmid pES1.

B. M13mp8 EP1-clone of 3'-end of cry gene

Plasmid pES1 was digested with PvuII and EcoRI and the resulting 2 kbfragment was isolated by agarose gel electrophoresis, Maniatis et al.supra 1982 p. 164 with the single exception that 1/10×TEA (4 mM Tris.0.1 mM Na EDTA 2H₂ O, 0.5 mM sodium acetate) was the buffer used. Thisfragment is from the 3'-end of the crystal protein gene and includes thetranscription termination signal of the crystal gene. The map locationand a portion of the sequence of the fragment, including the translationtermination codon to just after the transcription termination signal, isshown in FIG. 1.

Using conventional ligation methods, the 2 kb fragment was then clonedinto Replicative Form (RF) DNA of phage M13mp8 (available from BethesdaResearch Laboratories Inc., Gaithersberg, Md., USA) that had beenpreviously digested with SmaI and EcoRI. The resulting plasmid isdesignated M13mp8 EP1. E. coli strain JM103 was transformed with M13mp8EP1 plasmid.

C. M13mp9 NP3-subclone of 3'-end of cry gene

M13mp9 NP3 was prepared as follows: M13mp8 EP1 RF DNA was digested withNdeI. The NdeI ends were made blunt ended with E. coli DNA polymerase IKlenow fragment and dNTPs. The blunt ended DNA molecules were furtherdigested with BamHI. A 382 base pair (bp) fragment designated NP3carrying the transcription termination signal of the cry gene was thenisolated by acrylamide gel electrophoresis using the method described inMaxam et al., Proc. Natl. Acad. Sci. USA, 74:560-564 (1979). NP3 wassubcloned into a BamHI-SmaI digested M13mp9 RF DNA to form M13mp9 NP3.

E. coli JM103 was transformed with M13mp9 NP3.

The fragment designed NP3 cloned into M13mp9 NP3 is shown in FIG. 1. Ascan be seen in the figure, the fragment cloned into M13mp9 NP3 lacksapproximately 1.6 kb of DNA present between the NdeI and EcoRI sitesdownstream of the transcription termination signal in M13mp8 EP1.

EXAMPLE II Synthetic Genes Comprising Positive Retroregulatory Elements

A. pSYC667

Plasmid pSYC667 is capable of replication in E. coli and B. subtilis(see FIG. 3). It contains the gene for prepenicillinase (penP) from B.licheniformis 749/C which is expressed under the influence of aspecifically mutated signal sequence for penP in E. coli and B. subtilisthat have been transformed with the plasmid. Mature penicillinase issecreted extracellularly into the medium by B. subtilis transformed withpSYC667. Mature penicillinase is secreted into the periplasmic space ofE. coli transformed with pSYC667 and can be released from theperiplasmic space by osmotic shock according to the method of Nosel andHeppel, J. Biol. Chem., 241:3055-3062 (1966) or Lunn and Pigiet, J.Biol. Chem., 257:11424-11430 (1962).

pSYC667 was constructed as follows:

B. licheniformis penicillinase gene has been sequenced. See Kroyer, J.,and Chang, S., Gene, 15:343-347 (1981), and Neugebauer, K., et al.,Nucl. Acids Res., 9:2577-2588 (1981). The native signal sequencecontains the codon TGC which codes for a cysteine at amino acid position27. The cysteine residue at position 27 is modified as part of asequence of events leading to formation of the membranebound lipoproteinform of penicillinase. See Nielsen, J. B. K., Caulfield, M. P., andLampen, J. O., Proc. Natl. Aca. Sci. (USA), 78:3511-3515 (1981), andLai, J. S., Sarvas, M., Brammar, W. J., Neugebauer, K., and Wu, H. C.,Proc. Natl. Aca. Sci. (USA), 78:3506-3510 (1981). To specifically alterthis biosynthetic pathway, and shunt more of the protein to theextracellular form secreted from the cell (in the case of Gram positivebacteria such as B. subtilis) or into the periplasmic space (in the caseof Gram negative bacteria such as E. coli), it is necessary to mutatethe sequence in the penicillinase signal sequence gene coding forcysteine at this position.

Because of its simplicity and efficiency, the method of primer-directedmutagenesis (see Zoller, M. J., and Smith, M., Nucl. Acids Res.,10:6487-6500 (1982)) was used for the construction of the cysteine toserine mutation. A DNA fragment containing wild-type penicillinase(penP) gene sequence was isolated. Specifically the DNA fragment locatedbetween the HindIII and BamHI sites was excised from plasmid pSYC310-2.See McLaughlin, et al. Nucl. Acids Res., 3905-3919 (1982). PlasmidpSYC310-2 is capable of replicating in both B. subtilis and E. coli. Itcarries the wild-type penP gene from B. licheniformis 749/C on theHindIII-BamHI fragment. Those skilled in the art will realize that thewild-type penP gene could have been excised from other engineeredrecombinant plasmids that carry it. One such plasmid is B. subtilisplasmid pOG2165. The excised HindIII-BamHI DNA fragment from pSYC310-2was purified by acrylamide gel elution and then ligated to RF DNA ofcoliphage M13mp9. See Viera, J., and Messing, J., Gene, 19:259-268(1982) and Messing, J., and Viera, J., Gene, 19:269-276 (1982).

Specifically the purified HindIII-BamHI fragment from pSYC310-2 wasligated to M13mp9, obtained from Bethesda Research Laboratories, Inc.,P.O. Box 577, Gaithersburg, Md., that had previously been digested withrestriction endonucleases HindIII and BamHI. The double-stranded phageDNA was transformed into E. coli JM103, and the cells were cultured. Aclone transformed with recombinant phage carrying the penP gene, i.e.,recombinant phage M13-CM1, was identified and single-stranded phage DNAwas prepared from this clone. The methods used are described in Zoller,M. J., and Smith, M., Nucleic Acids Res., 10:6487-6500 (1982).

A 15-nucleotide synthetic fragment 5'-GTTAGCGGATCCTGC-3', made by thephosphotriester method of Narang, S. A., Hsiung, H. M., and Brousseau,R., in Methods in Enzymology, 68:90-97 (R. Wu, editor) Academic Press(1979), was first phosphorylated at the 5'-end with ATP and T4polynucleotide kinase and then employed as a primer to initiate thesynthesis of the complementary strand in vitro after the5'-phosphorylated primer had been annealed to the template M13-CM1 DNA.The primer was extended using DNA polymerase I Klenow fragment with allfour dNTP's in the presence of T4 ligase. This primer complements theanti-sense strand of the penP signal sequence gene segment correspondingto the codons for the five amino acids from positions 25 to 29, exceptthat the middle nucleotide in the synthetic primer is a mismatchednucleotide, that does not complement the corresponding nucleotide, G, inthe wild-type penP gene template. Incorporation of the mismatchedsequence into the penP gene causes conversion of the cysteine (TGCcodon) to serine (TCC codon) at position 27.

The alteration on the encoded peptide is essentially a conversion of the--SH group on the cysteine₂₇ to the --OH group of the serine₂₇. At thenucelotide level, a mutant gains a BamHI site (GGATCC) and loses theHhaI (GCGC) at the mutation locus. The presence of a new BamHI site wasthe phenotype used to identify the mutants carrying the "G to C"nucleotide mutation.

In constructing the cysteine to serine mutation, complementary (minus)strands were synthesized by primer-extension reaction using Klenowfragment of E. coli DNA polymerase I on the M13-CM1 phage DNA template.See Zoller, M. J., and Smith, M., Nucl. Acids Res., 10:6487-6500 (1982).In the presence of T4-DNA ligase in this reaction, a fraction of the DNAmolecules was converted to doublestranded, covalently-closed relaxedcircles. These molecules were separated from other molecules, whicheither were incompletely extended by polymerase or failed to be ligateddue to the incomplete kinase reaction of the primer. Separation wasaccomplished by agarose gel electrophoresis. This was carried out byapplying the reaction mixture on a 0.8% agarose gel in the presence of 2micrograms/ml of ethidium bromide. The band containing covalently closedcircular DNA was excised and DNA recovered.

Plasmid pSYC667 is the same as pSYC660 except that, in place of theBamHI recognition sequence (5'-GGATCC-3') at the end of theapproximately 1320 bp HindIII-BamHI fragment in pSYC660, pSYC667 has thesequence 5'-GGATCGATCC-3'.

Plasmid pSYC667 retains the PstI and BglII recognition sites of plasmidpSYC660. Similarly, the HindIII-BamHI fragment of M13penPS₂₇ P₂₈ thatcontains the penPS₂₇ P₂₈ gene has a PstI site and BglII site at the samelocations as the PstI site and BglII site, respectively, in theHindIII-BamHI-penPS₂₇ -containing fragment in pSYC667.

pSYC667 has been deposited at the ATCC under the terms of the BudapestTreaty and has been assigned ATCC No. 39758.

B. pHCW3-A3

Plasmid pSYC667 was digested with BclI, which cuts between thetranslation termination codon and transcription termination signal inthe B. licheniformis penicillinase gene. The BclI ends were filled inusing DNA polymerase I Klenow fragment and the four dNTP's. Such methodsare well known to those skilled in the art (see Maniatis, et al., surpa(1982)). Enzymes were inactivated by phenol extraction and the DNA wasrecovered by ethanol precipitation.

The DNA was then further digested with NruI, and extracted twice withphenol and then twice with ether. NruI cuts downstream of thetranscription termination signal of the B. licheniformis penicillinasegene in pSYC667. Thus, the large NruI-BclI cleaved fragment of pSYC667contains the entire penicillinase gene through the translationtermination codon but not including the transcription termination signal(see FIG. 3).

M13mp9 NP3 was cut with EcoRI and BamHI and a 400 bp fragment wasisolated with standard methods using a 1% agarose gel and purified withDE-52 chromatography. Such methods are described in Maniatis, et al.supra, 104 (1982).

The EcoRI-BamHI 400 bp fragment from M13mp9 NP3 was then ligated intothe large NruI-BclI fragment of pSYC667 using standard procedures. Theresulting plasmids were transformed into E. coli K12/CS412 using themethod of Cohen et al. (Proc. Natl. Acad. Sci. (USA), 69:2110 (1973);see also Maniatis et al. supra, 1982, p. 250). Transformants wereselected for resistance on LB medium plates containing 50 μg/mlampicillin. Maniatis et al. supra, 440 (1982). Plasmid DNA was preparedfrom selected transformants by the mini-prep method of Birnboim and Doly(Nucl. Acids Res., 7:1513 (1979)) and was screened for a plasmid withthe expected 1.6 kb EcoRI fragment, approximately 0.4 kb of the fragmentderived from M13mp9 NP3 and approximately 1.2 kb from pSYC667. Onetransformant harbors a plasmid which was designated pHCW-A3.

B.l. Transformation of B. subtilis with pHCW-A3

pHCW-A3 was also transformed into B. subtilis, PSL1, Bacillus GeneticStock Center No. IA510 (Bacillus Genetic Stock Center, Department ofMicrobiology, Ohio State University, Columbus, Ohio, U.S.A.). B.subtilis PSL1 is Leu⁻, Arg⁻, Thr⁻ and recE4.

A culture of B. subtilis PSL1 cells was made competent fortransformation using a technique related to that described byAnagnostopoulos and Spizizen, J. Bacteriol., 741-746 (1961).

10×Spizizen I Minimal Solution was prepared by mixing, in a totalsolution volume of 1 liter made up with distilled water, 20 gm (NH₄)₂SO₄, 140 gm K₂ HPO₄, 60 gm KH₂ PO₄ and 10 gm Na citrate.

Spizizen I Medium was prepared by mixing 2.05 ml of 1M MgSO₄ ; 6 ml of50% (w/w) glucose; 5 ml of 10% (w/w) yeast extract; 5 ml of 2% (w/w)casein hydrolystate; for each amino acid required by the strain to betransformed, 2.5 ml of 1% (w/w) solution of the amino acid; 50 ml of10×Spizizen I Minimal Solution; and enough distilled water to bring thesolution volume to 500 ml. For B. subtilis PSL1, the required aminoacids are threonine, arginine and leucine.

Spizizen II Medium was prepared by adding 0.25 ml of 1M CaCl₂ and 1 mlof 1M MgCl₂ to 500 ml of Spizizen I Medium.

30 ml of Spizizen I Medium was inoculated with a colony of spores of theB. subtilis strain to be transformed and was grown overnight (16-20hours) at 37° C.

15 ml of the overnight culture were then inoculated into 135 ml ofSpizizen I Medium in a 2800 ml flask and grown at 37° C. The opticaldensity at 600 nm (O.D.) of the culture was measured after 1.5 to 2hours, and then every 15 minutes until the culture was found to be inlate log phase on the basis of an increase in O.D. of less than 5%between 15-minute O.D. readings.

50 ml of late log phase culture was then inoculated into 450 ml ofSpizizen II Medium in a 2800 ml flask and grown at 37° C. for 1.5 hours.After the 1.5 hour growth, cells were spun down by centrifugation at5000 rpm for 10 minutes at 4° C.

The pellet from centrifugation was then resuspended in 45 ml ofsupernatant, to which 6 ml of 80% (v/v) sterile glycerol was then added,just prior to freezing the culture in a dry ice-ethanol bath (-70° C.).The cells in the frozen culture were competent cells, suitable fortransformation by the desired plasmid, as follows:

0.5 ml-0.6 ml of the frozen, competent-cell-containing culture wasthawed on ice, and 5 microliter to 50 microliter of solution containingthe plasmid to be transformed into the cells was combined with thethawed culture. The resulting mixture was shaken at 37° C. for 2 hours,during which transformation of plasmids and expression of genes on themoccurred.

Finally, for selection, small aliquots such as about 5 microliters toabout 200 microliters of the culture of transformed cells weretransferred to plates containing the desired antibiotic or antibioticsfor selection.

A mini-prep of plasmid pHCW-A3 was prepared as described above from E.coli K-12/CS4-12 transformed with the plasmid. To 1 ml of culture ofcompetent B. subtilis PSL1, 5 microliters of mini-prep plasmid DNAsolution was added. The mixture was incubated at 37° C. for 2 hours.Aliquots of the mixture were then plated and incubated at 37° C.overnight on rich medium agar plates (beef extract, 1.5 g/liter; yeastextract, 3.0 g/liter; peptone, 6.0 g/liter; agar 15.0 g/liter) to which5 micrograms/ml chloramphenicol had been added. Achloramphenicol-resistant colony was picked and inoculated into 5 ml of2×LB medium containing 5 μg/ml chloramphenicol; the culture wasincubated overnight at 37° C. with shaking.

Subcultures of cultures of B. subtilis PSL1 transformed with pHCW-A3,and prepared as above, have been deposited in the CMCC under collectionnumber 2120.

C. pLW1

Plasmid pLW1 is a pBR322 derivative capable of replication in E. colicontaining a tetracycline resistance gene the E. coli trp promoter,ribosome binding site (RBS) fragment and a 706 bp HindIII-PstI DNAfragment which includes the gene for human interleukin-2 (IL-2)(Rosenberg, S. A. et al. Science,223:1412-1415 (1984)). pLW1 has beendeposited at the ATCC under terms of the Budapest Treaty and assignedATCC No. 39405.

D. pLW45

Plasmid pLW45 is a pBR322 derivative capable of replication in E. colicontaining a tetracycline resistance gene and the E. coli trp promoter.The plasmid contains, on a 706 bp HindIII-PstI fragment, a gene for amodified IL-2 protein.

pLW45 has been deposited at the ATCC under terms of the Budapest Treatyand assigned ATCC No. 39629.

The modified IL-2 it encoded by pLW45 and the uses of such modified IL-2protein in treating human diseases involving suppression of the immunesystem are described in Belgian Pat. Ser. No. 898,016, issued Nov. 14,1983, which is incorporated herein by reference.

E. pHCW701 and pHCW702

The 400 bp EcoRI-BamHI restriction fragment carrying the transcriptiontermination signal of the cry gene was excised from M13mp90 NP3 bydigestion with EcoRI and BamHI restriction endonucleases under bufferconditions suggested by the supplier. The EcoRI-BamHI ends of thefragment were made blunt ended with Klenow PolI fragment and dNTPs. Theblunt ended fragment containing the transcription terminal signal of thecry gene was isolated by acrylamide gel electrophoresis. The isolatedblunt ended fragment was electroeluted and ligated using T4 ligase andATP into plasmid pLW1 that had been previously digested with StuIrestriction endonuclease.

Regardless of the orientation in which the blunt ended fragment carryingthe transcription termination signal is recombined with the StuI ends ofpLW1, both of the original EcoRI and BamHI sites will be regenerated.One orientation results in the BamHI site being located nearer to the 3'end of the IL-2 gene and the plasmid so characterized is designatedpHCW701. (This orientation is similar to that found in the cry geneitself.) The other orientation results in the EcoRI site being nearer tothe 3' end of the IL-2 gene and this recombinant plasmid is designatedpHCW702 (FIG. 3). Due to the a symmetrical location of the invertedrepeat sequence in the EcoRI-BamHI fragment as depicted in FIG. 1, thestem and loop structure of the positive retroregulatory element islocated approximately 310 bp downstream of the BamHI site at the 3' endof the IL-2 gene in pHCW701. In pHCW702, the stem and loop structure ofthe positive retroregulatory element is approximately 30 bp downstreamof the EcoRI site of the 3' end of the IL-2 gene.

pHCW701 and pHCW702 can be easily distinguished by digesting the plasmidDNAs with restriction enzyme EcoRI and determining the size ofrestriction fragments using acrylamide or agarose gel electrophoresis.EcoRI digested pHCW701 releases a 960-bp restriction fragment whichcontains the trp promoter-RBS cassette (108 bp), the IL-2 gene (450 bp)and the terminator (400 bp). However, EcoRI digested pHCW702 releases a560-bp restriction fragment which contains only the trp promoter-RBScassette (108 bp) and the IL-2 gene (450 bp).

F. Construction of pHCW801

pHCW801 was constructed to assess the effect of the positiveretroregulatory element on productin of the modified IL-2 by plasmidpLW45. Plasmid pHCW701 was digested with BamHI and the ends were madeblunt with Klenow PolI fragment and dNTPs as previously described. Theblunt ended BamHI fragment was digested with AvaI. The largest resultingfragment was 2.7 kb and had one blunt BamHI end and one AvaI end. Thisfragment was purified by 0.8% agarose gel electrophoresis, and containsthe 3' expression enhancement sequence.

Plasmid pLW45 was digested with StuI and then AvaI restrictionendonucleases. An approximately 2.3 kb fragment was purified from thedigest by agarose gel electrophoresis, electroeluted, and ligated usingT4 ligase and ATP, to the fragment having a BamHI blunt end and an AvaIend, derived from plasmid pHCW701, which contained the positiveretroregulatory element. Tetracycline resistant transformants wereanalyzed by mini-prep isolation of plasmid DNA and screened for thepresence of an EcoRI fragment including both the modified IL-2 gene andthe retroregulatory element. Birnboim and Doly, supra (1979).

G. pHCW301

1. Promoter 156:

A promoter, recognized by B. subtilis vegetative RNA polymerase, whichis located on a 240 bp HhaI restriction fragment of the bacteriophageSP82, was first discovered by DNA restriction fragment probe analysesand B. subtilis RNA polymerase binding and initiation assay. Jones, B.B., Chan, H., Rothstein, S.; Wells, R. D. and Reznikoff, W. S., Proc.Natl. Acad. Sci. (USA), 74:4914-4918 (1979). The 240 bp HhaI fragmentwas isolated from the HhaI digested SP82 DNA by gel electrophoresis andthe ends were made blunt by removal of unpaired nucleotides with Slnuclease (2200/ml in pH 4.6 buffer containing 300 mM NaCl, 60 mM SO₄ and50 mM Na acetate). Maniatis et al. supra at p. 140. The blunt endfragment was then cloned into the HincII site of M13mp7 RF DNA (obtainedfrom Bethesda Research Labs) that had been previously digested withHincII restriction endonuclease. DNA sequence analysis indicated that aRBS sequence was located at the 3' end of the HhaI restriction fragmentcarrying the promoter sequence. To eliminate the RBS sequence, the 240bp HhaI fragment was digested with AluI restriction endonuclease. A 150bp HhaI-AluI restriction fragment was isolated by acrylamide gelelectrophoresis. The promoter was known to be located on this 150 bprestriction fragment by DNA sequence analysis. The 150 bp restrictionfragment was then subcloned into the HincII site of M13mp7 RF DNA whichhad been previously digested with HincII restriction endonuclease toform a phage designated M13mp7-p156.

2. The ribosome binding site:

Two synthetic oligonucleotides with the sequence: (1)5'-CGATAAGAGGAGGTA-3' and (2) 5'-AGCTTACCTCCTCTTAT-3' were made.

500 picomoles of each oligonucleotide were mixed and phosphorylated withpolynucleotide kinase and ATP. The phosphorylated oligonucleotides werethen annealed at 68° C. for 1 hour and then at 37° C. for 3 hours. Theannealed oligomer having the sequence ##STR20## was then cloned into theClaI-HindIII site of pUC8-41 using T4 ligase under ligation conditionsat a molar ratio of the oligomer to vector of approximately 10 to 1.pUC8 is commercially available (Bethesda Research Laboratories,Gaithersberg, Md. USA) and was modified as follows to yield pUC8-41:pUC8 was digested with BamHI and the ends were made blunt with KlenowPolI fragment. White colonies of E. coli JM103 containing therecircularized BamHI repaired pUC8 were selected. Plasmid DNA from thesetransformants was linearized with PstI and the ends were trimmed withKlenow PolI fragment. This procedure restores the correct reading framefor lac Z and after ligation blue transformants of JM103 were selected.Since repair of the BamHI site generates a ClaI site in DNA preparedfrom a DNA methylase lacking (Dam⁻) E. coli host, pUC8-41 was confirmedby ClaI linearization. Restriction enzyme analysis and nucleotidesequence determination were used to determine that the constructedrecombinant plasmid designated pUC8-41-RBS 1 was indeed carrying asingle copy of the synthetic ribosome binding site.

3. Construction of pLP1201-p156-RBS1

The synthetic ribosome binding site in pUC8-41-RBS1 was recovered byEcoRI-HindIII double digestion of the plasmid and was isolated byacrylamide gel electrophoresis. The EcoRI-HindIII fragment was subclonedinto plasmid pLP1201 that had been previously digested with EcoRI andHindIII restriction endonucleases to form pLP1201-RBS1. Ostroff, G. R.and Pene, J. M. Mol. Gen. Genet., 193:306-311 (1984). E. coli strainCS412 was transformed with plasmid pLP1201-RBS1 using conventionalmethods. Cohen et al. supra (1973). E. coli transformants carrying theplasmid pLP1201-RBS1 were ampicillin resistant and tetracyclinesensitive.

Promoter p156 was then excised from M13mp7-p156 RF DNA using EcoRIrestriction endonuclease and subcloned into the EcoRI site ofpLP1201-RBS1 that had been previously digested with EcoRI restrictionendonuclease. The desired recombinant plasmid pLP1201-p156-RBS1 confersampicillin and tetracycline resistance to the E. coli host CS412transformed with the plasmid.

4. Construction of plasmid pHCW300 (pLP1201-RBS1-p156-IL-2)

pHCW300 was constructed from pLW21 and pLP1201-RBS1-p156. pLW21 isderived from pBR322 and contains a 570 bp EcoRI-BanII sequence includinga region coding for IL-2. pLW21 was constructed by ligating the 570 bpEcoRI-BanII sequence, obtained by digesting pLW1 with EcoRI and BanIIendonucleases, into pBR322 previously digested to completion with EcoRIand BanII. This EcoRI-BanII fragment replaces the 485 bp EcoRI-BanIIfragment in pBR322 containing a portion of the tetracycline resistancegene to yield pLW21 which is tetracycline sensitive. pLW21 was digestedwith HindIII restriction endonuclease followed by digestion with NruIrestriction endonuclease. Two fragments were generated and aHindIII-NruI fragment approximately 400 bp in length was isolated byacrylamide gel electrophoresis. Plasmid pLP1201-RBS1-p156 was digestedwith HindIII and NruI restriction endonuclease to form a linearizedplasmid with the region containing the tetracycline resistance generemoved. The linearized plasmid was combined with the 400 bpHindIII-NruI fragment from pLW21 and was ligated using T4 ligase to forma plasmid designated pHCW300 having the p156 promoter and RBS describedabove and a 400 bp sequence having a DNA sequence coding for IL-2.Tetracycline sensitive transformants of E. coli were obtained by replicaplating of ampicillin resistance transformants and were screened for thepresence of plasmid pHCW300 using HindIII and AvaI restrictionendonucleases to generate two fragments of about 7 kb and 0.85 kb.

5. Construction of plasmid pHCW301(pLP1201-RBS1-p156-IL-2-retroregulator)

Plasmid pHCW701 was digested with EcoRI restriction endonuclease and theends were made blunt with Klenow Pol I fragment and dNTPs. Followingdigestion with HindIII restriction endonuclease, the approximately 0.85kb fragment containing the IL-2 gene and the positive retroregulatoryelement was isolated by agarose gel electrophoresis. PlasmidpLP1201-RBS1-p156 was digested with HindIII and NruI restrictionendonucleases resulting in the excision of the region containing thetetracycline resistance gene. The 0.85 kb EcoRI (blunt)-HindIII fragmentcontaining the IL-2 gene and the positive retroregulatory element wasligated to the HindIII-NruI digested pLP1201-RBS1-p156 vector using T4ligase under ligating conditions. Tetracycline sensitive transformantsof E. coli were obtained by replica plating of ampicillin resistanttransformants and were screened for the plasmid pHCW301 using EcoRIrestriction endonuclease. Three fragments of approximately 7 kb, 0.88kb, and 156 bp corresponding to the expected sizes for thepLP1201-RBS1-fragment, IL-2 gene-positive retroregulatory elementfragment, and promoter fragment respectively, were generated.

EXAMPLE III Enhanced Protein Expression by Genes into Which a PositiveRetroregulatory Element Has Been Inserted

A. Assay procedure for B. licheniformis penicillinase expression

1. In E. coli

Five ml of YT broth (8 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl)containing 50 μg/ml ampicillin were inoculated with individual coloniesof E. coli K-12/CS412 carrying either pSYC667 or pHCW-A3 and were grownovernight at 37° C. with shaking in a New Brunswick rotary incubator.Cells were then pelleted at 5,000 rpm for 10 minutes in the JA-20 rotorof a Beckman Model J-21C centrifuge. The cell pellet was washed with 5ml ice-cold 50 mM phosphate buffer pH7.0. The washed cell pellet wasresuspended into 0.5 ml 50 mM phosphate buffer and sonicated using aBranson 350 Sonifier at maximum output for 3 minutes in 0.5 secondbursts at 0° C. The sonicate was spun in an Eppendorf microfuge for 2minutes. The resulting supernatant was used for the assay of enzymeactivity.

The production of B. licheniformis penicillinase was assayed using thechromogenic β-lactamase substrate PADAC (available from Cal Biochem) bythe method of Schindler and Huber. Schindler, P. and Huber, G., "Use ofPADAC, A Novel Chromogenic, β-Lactamase Substrate, for the Detection ofβ-Lactamase Producing Organisms and Assay of β-LactamaseInhibitors/Inactivators", in Enzyme Inhibitors, Brodbeck, U., ed.,Weinheim:VerlagChemice, 1980, p. 169-176. PADAC substrate (MW 562.7) wasprepared by making a solution having OD₅₇₃ =1 (about 27.4 μM) inphosphate buffer at pH 7.0. The decrease in absorbance at 573 nm overtime, after the addition of the cell extract, was measured using a Cary219 spectrophotometer at room temperature. The results are shown inTable I.

2. In B. subtilis

B. subtilis PSL1 was transformed with either pSYC667 or pHCW-A3,according to the method related to Anagnostopolus and Spizizen supra(1961) described above, plated on rich medium agar described above, andgrown overnight at 37° C. Five ml of 2×LB medium containing 5 μg/mlchloramphenicol was inoculated with individual transformants and grownat 37° C. with shaking on a New Brunswick rotary incubator overnight.Cells were then pelleted at 5000 rpm for 10 minutes in a JA-20 rotor asabove. The supernatant was used for the penicillinase assay by the samemethod described above for E. coli. The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Production of the penicillinase polypeptide in E. coli                        and B. subtilis strains carrying penP recombinant                             plasmids with or without the positive                                         retroregulatory element                                                                               Specific Activity of                                  Host          Plasmid   Penicillinase.sup.(1)                                 ______________________________________                                        E. coli CS412 --        0                                                                   pSYC667   1055                                                                pHCW-A3   2762                                                  B. subtilis 1A510                                                                           --        0                                                                   pSYC667   3280                                                                pHCW-A3   17510                                                 ______________________________________                                         .sup.(1) Specific activity is defined as nmoles of PADAC hydrolyzed per       minute per mg of protein at 25° C.                                

B. Assay procedure for IL-2 expression

1. In E coli

A shake flask culture of E. coli K12/MM294 transformed with pHCW701,pHCW702, or pLW1 were grown in 10 ml of tryptophan (trp) containing Nmedium (0.7% Na₂ HPO₄, 0.3% KH₂ PO₄, 0.5% NaCl, 0.1% NH₄ Cl, 0.2%glucose, 0.5% casamino acids, 40 μg/ml trp, and 10 μg/ml tetracycline)at 37° C. overnight with shaking in a New Brunswick rotary incubator.Cells from a 5 ml overnight culture were pelleted by centrifugation at5000 rpm for 10 minutes in a JA-20 rotor. The cell pellet wasresuspended in 5 ml of N medium without trp containing 2 μg/ml thiamine.Optical density was determined by absorbance at 600 nanometers (OD₆₀₀)in a spectrophotometer. 25 ml subcultures having an OD₆₀₀ of 0.05 in Nmedium minus trp were set up and grown at 37° C. with shaking to a finalOD₆₀₀ of about 0.3. Controls were set up and grown under the sameconditions except that the N medium lacked tryptophan. Cells were thenpelleted and resuspended in IL-2 sonication buffer (50 mM Tris, pH 7.5,50 mM EDTA, 15% sucrose, 1% SDS) to a final OD₆₀₀ =10. Cells weresonicated as described above. Supernatants were assayed for the presenceof IL-2 activity by the methods described in Gillis, S., et al. J.Immunol., 120, 2027-2032 (1978). Results are shown in Table II.

2. Fermentation cultures

E. coli K-12/MM294-1 transformed with either pHCW801 or pLW45 wasfermented in a 10 liter fermentor at 37° C. and 350-1200 rpm with 0-2liters per minute (lpm) air and 0-5 lpm oxygen (dissolved oxygen atabout 40%). The medium consisted of 72 mM (NH₄)₂ SO₄, 21.6 mM KH₂ PO₄,1.5 mM Na₃, citrate, 1.5 mg/l TK-9 Trace elements, and the followingsterile additions: 3 mM 0.5% MgSO₄, 20 mg/l 1% thiamine-HCl, 72 mM 0.2MFeSO₄, 5 g/l 50% glucose, 70 mg/l 0.5% L-tryptophan, 5 mg/l 1%tetracycline, and 100 ml/l 20% casamino acids (added at OD₆₈₀ =15-20).The inoculum was 20 mg/l and the pH was controlled at 6.8 with 5N KOH. Aglucose feed was also employed to maintain glucose concentration between5-10 g/l.

Culture samples for SDS polyacrylamide gel electrophoretic analysis oftotal cell protein were withdrawn hourly from 13.7 hours to 19.7 hours.Densitometry of protein bands of the gels indicated a maximum productionof IL-2 as 17.2% of total cell protein at 17.7 hours. The biologicalactivity of IL-2 from these samples was determined as previouslydescribed for IL-2 expression in E. coli. Results are shown in Table II.

3. In B. subtilis

Cultures of B. subtilis 1A510 transformed as described above withpHCW301 or pHCW300 were grown to an OD₆₀₀ =1.0 at 37° C. with shakingafter single colony inoculation into 5 ml of 2×LB medium containing 5μg/ml chloramphenicol. Cells were pelleted, resuspended in IL-2sonication buffer, sonicated and assayed for IL-2 activity, as describedfor E. coli. Results are shown in Table II.

                  TABLE II                                                        ______________________________________                                        Production of IL-2 and modified IL-2                                          with and without the positive                                                 retroregulatory element                                                       1. In E. coli (5 ml culture)                                                                      Specific Activity of IL-2 Produced                        Transformed with                                                                         Induction                                                                              (U/150 μg total Cellular Protein                       ______________________________________                                        pLW1       -        <7 × 10.sup.3                                                  +        2 × 10.sup.5                                        pHCW701    -        2.8 × 10.sup.4                                                 +        1.4 × 10.sup.6                                      pHCW702    -        1.5 × 10.sup.5                                                 +        9.2 × 10.sup.5                                      ______________________________________                                        2. In E. coli (10 1 Fermentor)                                                Transformed with                                                                              IL-2 Produced (U/g dry weight)                                ______________________________________                                        pLW45           8.95 × 10.sup.7                                         pHCW801         1.6 × 10.sup.8                                          ______________________________________                                        3. In B. subtilis (5 ml culture)                                                              Specific Activity of IL-2 Produced                            Transformed with                                                                              (U/150 μg Total Cellular Protein)                          ______________________________________                                        pHCW300         1 × 10.sup.4                                            pHCW301         2.5 × 10.sup.4                                          ______________________________________                                    

EXAMPLE IV Deletion Mapping of the Positive Retrogulatory Element

Deletion studies were carried out to define more precisely the sequencewithin the cry terminator region responsible for the positiveretroregulatory effect. Oligodeoxyribonucleotide-directed site-specificmutagenesis according to the method of Zoller, M. and Smith, M.,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors Methods in Enzymology, 100:468-500 (1983) was employed tointroduce two BglII restriction sites separately at the locations 80-and 20-bp upstream from the inverted repeat sequence in the cryterminator fragment depicted respectively as B1 and B2 in FIG. 1. Theseshortened terminator-containing fragments can be excised from therespective, modified M13mp9 NP3 phage genomes by BglII-EcoRI digestion.They were cloned into pSYC667 at the BclI-NruI site by the proceduressimilar to that employed for the construction of pHCW-A3, and generatedplasmids pHCW-A4 and pHCW-A5. Data on the analysis of B. subtilis aswell as E. coli strains harboring these plasmids for their ability toexpress the cloned penP gene are presented in Table III. It is clearthat the shortened fragments still contain the positive retroregulatoryelement observed in the original fragment. Since the two newly createdBglII sites are outside of the cry coding sequence, this datademonstrates that the locus that confers the enhancing activity islocated in the non-coding region of the cry gene, and it probablyoverlaps with the terminator of cry gene.

B. subtilis strain PSL1 and E. coli strain CS412 were transformed withplasmids pHCW-A3, pHCW-A4, or pHCW-A5 as described above. Penicillinaseactivity, assayed as described above is shown in Table III.

                  TABLE III                                                       ______________________________________                                        Synthesis of Penicillinase in E. coli                                         and B. subtilis Strains Carrying the                                          penP-cry Fusion Plasmids.sup.(1)                                                                   Length of                                                                     cry-derived                                                                              Specific Activity                             Host Strain                                                                              Plasmid   Fragments  of Penicillinase.sup.(1)                      ______________________________________                                        E. coli CS412                                                                            pHCW-A3   380         2762                                                    pHCW-A4   158         2631                                                    pHCW-A5    78         2861                                         B. subtilis 1A510                                                                        pHCW-A3   380        17510                                                    pHCW-A4   158        18600                                                    pHCW-A5    78        17822                                         ______________________________________                                         .sup.(1) Penicillinase was assayed as described in Table I.              

EXAMPLE V Insertion of Positive Retroregulatory Element Increases theLevel of Production of a Novel Recombinant IL-2 Mutein using a PortableTemperature Regulated Control Cassette in a Temperature Regulated ColE1Copy Mutant Plasmid Vector

A. Construction of plasmid pFC54.t

Plasmid pFC54 encodes des-Alanyl, serine₁₂₅ interleukin-2 (IL-2) underthe control of the bacteriophage λ P_(L) promoter and gene N ribosomebinding site (P_(L) N_(RBS)). The ColE1 plasmid vector contains twomutations which confer a temperature sensitive copy number phenotype onthe plasmid. E. coli cells harboring this plasmid have been thermallyinduced to accumulate 20% of the total protein as a novel IL-2 mutein.

Plasmid pFC54 was digested to completion with SphI and treated with DNApolymerase I (Klenow fragment) in the presence of 25 μM dGTP toeliminate the 3' protruding single stranded tail. The blunt-ended DNAwas subsequently digested with XbaI.

Plasmid pHCW801 was digested to completion with EcoRI, treated with DNApolymerase I (Klenow fragment) in the presence of dNTPs, andsubsequently digested with XbaI. The 655 bp DNA fragment comprising theC-terminal 225 bp of ser¹²⁵ IL-2, 36 bp of 3' untranslated IL-2 cDNA and394 bp of the fragment carrying the positive retroregulatory elementwere purified by gel electrophoresis. The pFC54 vector DNA fragments andpurified pHCW801 fragment were ligated (1:3 molar ratio) at aconcentration of 30 μgDNA/ml DNA using T4 DNA ligase under conditionsfavoring ligation of sticky ends, diluted 2.5 fold and then ligatedusing T4 DNA ligase under blunt-end ligation conditions to favorintramolecular circle formation. The ligated DNA was digested with BanIIto inactivate undesired ligation products comprised of the small andlarge XbaI-SphI fragments of pFC54.

E. coli K12 strain DG95(λN₇ N₅₃ cI857susP80) was transformed to Amp®with 60 ng of the ligated and digested DNA. This strain contains alambda prophage which encodes a temperature-sensitive λ cI repressor,which at low temperature (30°-32° C.) is active. However, at hightemperature (36°-42° C.) the repressor is inactive and transcriptionfrom the P_(L) promoter can proceed. It is further characteristic ofthis strain that at elevated temperatures the prophage fails to induce.Transformants were selected for Amp® and Amp® colonies were screened forthe desired 5.6 kb plasmid. Candidate plasmids were screened for releaseof an 1182 bp EcoRI fragment (ligation of repaired EcoRI site in thefragment carrying the positive retroregulatory element to the repairedSphI site in the vector fragment was expected to regenerate an EcoRIsite), retention of the XbaI site within the IL-2 coding sequence,acquisition of a unique BamHI site (in the terminator fragment), andloss of the unique BanII site in pFC54. A desired recombinant plasmidwas isolated and was designated pFC54.t.

As shown below, when E. coli K12 strain DG95(λN₇ N₅₃ cI857susP80)harboring plasmid pFC54.t is temperature-induced under the properconditions, 34% of the total cellular protein is des-Alanyl, Ser¹²⁵IL-2.

Plasmid pFC54.t has been deposited pursuant to the Budapest Treaty inthe ATCC under accession number 39789.

Plasmid pFC54.t is shown in FIG. 6. Beginning with the EcoRI restrictionendonuclease site designed 5.61/0 and moving in a clockwise direction,plasmid pFC54.t comprises the components described immediately below.

Coordinate 0-0.35 kb comprise an EcoRI-HindIII module encoding thetemperature regulated promoter/operator of the bacteriophage λ PLpromoter and the adjacent gene N ribosome binding site. The BglIIrecognition site at λ coordinate 35715 (Sanger, F. et al., J. Mol.Biol., 162:729-773 (1982) has been converted to an EcoRI recognitionsite and the HinfI recognition site at λ coordinate 35366 has beenconverted to a HindIII recognition site for insertion into plasmidpFC54.t.

Coordinates 0.35-0.794 kb comprise the 444 bp HindIII-StuI fragment ofplasmid pLW46 encoding mature human des Alanyl, Ser¹²⁵ IL-2 mutein.Wang, A. et al., Science, 224:1431-1433 (1984).

The 5' HindIII site immediately precedes the ATG initiation codon of thealtered mutein (Rosenberg, S. A. et al. Science, 223:1412-1416 (1984))and the StuI recognition site (36 bp distal to the opal stop codon inhuman IL-2) has been converted to a BamHI recognition site in pFC54.t.

Coordinates 0.794-1.188 kb comprise the 394 bp BamHI-EcoRI DNA fragmentfrom plasmid pHCW801 and includes the positive retroregulatory elementfrom the B. thuringiensis delta endotoxin gene.

Coordinates 1.188-2.05 kb comprise the 863 bp SphI (repaired)/AvaI(repaired) pBR322 DNA fragment (pBR322 coordinates 567-1429,respectively).

Coordinates 2.05-3.48 kb comprise the 1.43 kb PuvII-BamHI DNA fragmentfrom plasmid pOP6 (Gelfand, D. H., et al., Proc. Natl. Acad. Sci. USA,75:5869-5873, 1978; Muesing, M., et al., Cell, 24:235-242, 1981)encoding the β-lactamase gene of bacterial transposon Tn3. Heffron, F.,et al., Cell, 18:1153-1163 (1979).

Coordinates 3.48-3.95 kb comprise the 0.47 kb SstI-PvuII DNA fragment ofpOP6 (Gelfand, supra 1978). This DNA fragment contains the left boundaryof bacterial transposon Tn3 (coordinate 1-270, Heffron, F., et al. supra1979) and a portion of the adjacent ColE1 HaeII C fragment.

Coordinates 3.95-5.61 kb comprise the 1.66 kb PvuII-EcoRI DNA fragmentfrom plasmid pEW27 (Wong, E. M., et al., Proc. Natl. Acad. Sci. USA,79:3570-3574 (1982)). This fragment encodes the colicin E1 immunityregion of plasmid ColE1 as well as the primer promoter region, RNA Iregion, and origin of replication. The wild-type ColE1 DNA sequence ofpBGP120 (Polisky, B., et al., Proc. Natl. Acad. Sci. USA, 73:3900-3904(1976) and Gelfand, D. H., et al., supra 1978) has been altered at twopositions (G→A transitions in the DNA strand corresponding tonucleotides 125 and 135 of the primer RNA transcript) conferring atemperature sensitive Cop-phenotype on the plasmid pFC54.t

B. IL-2 mutein produced by the transformed strains

E. coli K12 strain DG95 (λN₇ N₅₃ cI857susP80), transformed with eitherpFC54 or pFC54.t, using methods described above were grown in 10 literfermentors under the same conditions as described above for 18.5 hoursto an OD₆₈₀ of 28.7 or 28.1 respectively. Following measurement at 18.5hours, the temperature of the culture medium containing transformedmicroorganism was raised to 42° C. to induce the temperature sensitivecopy number plasmid and to inactivate the xeI represion and allowtranscription from the promoter. Samples were taken at half hourly orhourly intervals after induction for 4 hours. Each sample was pelletedby centrifugation and was resuspended to a concentration of 10 mg dryweight (dw)/ml in phosphate buffered saline (PBS). Each sample was thendiluted 10×in 1% sodium dodecyl sulphate/PBS, sonicated in a HeatSystems Model W-375 sonicator for a period sufficient to completelydisrupt the cells and assayed. Units of IL-2 mutein/ml was determined inthe manner described above. Mg protein/ml sonicate was determined by theLowry method. Units IL-2 mutein/mg protein was determined and isreported in Table IV below.

The production of IL-2 as a percentage of total cellular proteinproduced for each sample was determined by SDS acrylamide gelelectrophoresis. Approximately equal amounts of protein as determined bythe Lowry assay were loaded onto the gel. Bands were stained withCoomasie Blue strain and were read using a Zeineh scanning densitometerattached a Hewlett Packard 3390A integrator. Percent IL-2 mutein wasdetermined by the integration program. Il-2 mutein production as apercentage of total cellular protein is reported in Table V below.Percent increase in Il-2 was found by determining the net increase inIL-2 mutein produced by the strain carrying the positive retroregulatoryelement and expressing the increase as a percentage of the IL-2 muteinproduced by the strain without the positive retroregulatory element.

E. coli strain K12 DG95 (λN₇ N₅₃ cI857susP80) transformed with pFC54.twas grown under the same conditions as described above except that theculture was temperature induced at an OD₆₈₀ of 14.0 rather than 28.1 asin the previous example. Samples were taken and determinations were madeas in the previous example. IL-2 mutein production as a percentage oftotal cell protein was determined to be 34% of total cellular protein.Increased production of IL-2 mutein by the microorganism transformedwith the plasmid carrying the positive retroregulatory element is bestaccomplished when temperature induction is carried out at lower celldensity as measured by OD₆₈₀ (14) than higher OD₆₈₀ (28.1).

                  TABLE IV                                                        ______________________________________                                        U IL-2/mg Protein                                                             Time after                                                                             E. coli with                                                                             E. coli with                                                                             % Increase                                     Induction                                                                              pFC54      pFC54.t    U IL-2/mg Protein                              ______________________________________                                        0        715 × 10.sup.4                                                                     8.60 × 10.sup.4                                                                    10.3                                           1 hr.    1.44 × 10.sup.5                                                                    1.62 × 10.sup.5                                                                    12                                             1.5 hr.  1.99 × 10.sup.5                                                                    2.26 × 10.sup.5                                                                    13.5                                           2 hr.    2.54 × 10.sup.5                                                                    2.68 × 10.sup.5                                                                    5.5                                            3 hr.    2.23 × 10.sup.5                                                                    3.02 × 10.sup.5                                                                    26.1                                           4 hr.    2.85 × 10.sup.5                                                                    2.52 × 10.sup.5                                                                    --                                             ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        IL-2 Mutein                                                                   % Total Cellular Protein                                                      Time     pFC54       pFC54.t  % Increase                                      ______________________________________                                        0 hr.    3.3         2.6      --                                              0.5 hr.  12.3        12.7      2                                              1 hr.    14.7        17.9     21                                              2 hr.    24.3        21.5     --                                              3 hr.    21.6        20.6     --                                              4 hr.    23.8        22.4     --                                              ______________________________________                                    

EXAMPLE VI Half-life extension of mRNA transcripts by the positiveretroregulatory element

In order to determine whether the enhancement of the expression of theselected DNA sequence ligated to the positive retroregulatory element isthe result of increased stability and extended half-life of the mRNAtranscript encoded by the selected DNA sequence, the rates of decay ofthe penP transcripts produced by plasmid pHCW-A3 and its parentalplasmid pSYC667 in both E. coli and B. subtilis were measured. E. colistrain CS412 and B. subtilis strain 1A510 each carrying plasmid pHCW-A3or its parental plasmid pSSYC667, were grown in L-broth at 37° C. withshaking. When the culture reached mid-log phase of growth (A ₆₀₀ =0.7),1 mg/ml rifampicin was added to block further initiation oftranscription by RNA polymerase. Seven ml samples were then withdrawnfrom the culture at one minute intervals and the tubes containing thesamples were rapidly chilled on ice water. The cells were harvested bycentrifugation and resuspended into 500 μl potassium acetate buffer, pH5.5, containing 1 mM EDTA and 10% (w/v) SDS. Total cellular RNA from E.coli was extracted and purified as described in Gabain et al., Proc.Nat. Acad. Sci. USA, 80:653-657 (1983).

RNA preparations from B. subtilis were extracted by the followingprocedure: frozen cell pellets (1 g) were suspended in 4 ml of 0.05Mpotassium acetate, pH 5.0, containing 0.05 mg/ml of bentonite per ml,and 2 ml of freshly distilled phenol (saturated with the same potassiumacetate buffer) were added. The suspension was sonicated for threeminutes using a Branson Sonifier (Heat Systems-Ultrasonics, Inc.,Plainview, Long Island, N.Y.) equipped with a microtip, extracted at 60°C. for 10 minutes, and precipitated overnight with ethanol. Theprecipitated material was collected by centrifugation, washed with -20°C. ethanol, dissolved in 4 ml of 0.104M Tris, pH 7.0, 0.01M MgCl₂. DNAse(30 μg/ml, Worthington, RNAse free) was added. The preparation wasdialyzed for four hours at 37° C. against 400 volumes of 0.04M Tris, pH7.0, 0.01M MgCl₂. The RNA was then extracted twice with phenol at roomtemperature for five minutes, precipitated with ethanol overnight,washed with ethanol, and dissolved in water.

The purified mRNAs were immobilized on 82 mm nitrocellulose filters(grade BA 85, Schleicher and Schuell, Inc.) Prehybridization of thefilters was carried out by the procedure of Woo et al. as described inMethods in Enzymology, 68:389 (1979).

Processed filters were then hybridized with penP-specific probes (20μg/filter) in 10 ml hybridization buffer (5×Denhards solution, 50 mMsodium phosphate, pH 7.0, 100 μg/ml sheared single-stranded E. coli DNA,1% SDS) at 68° C. overnight.

The probes used in the hybridization were made by completely digestingplasmid pSYC795 with ClaI, and repairing the ends with dNTPs and DNApolymerase large fragment to form a linearized plasmid. Plasmid pSYC795is a derivative of plasmid pSYC423, which is described in Hyashi et al.,J. Biol. Chemistry, 259:10448-10454 (1984), which contains a G to Cmutation at nucleotide 80 of the coding region of the penP gene. Thelinearized plasmid was further digested with EcoRI. The plasmid digestwas run on a 5% polyacrylamide gel and a 727 bp EcoRI-ClaI fragment,containing the 5' portion of the penP gene was recovered. This fragmentwas subcloned into the SmaI-EcoRI site of M13mp11. Single-stranded DNAsfrom these recombinant phages were isolated according to the method ofMessing, Methods in Enzymology, Vol. 101:20-78 (1983), and the circularDNA was digested with DNAse I (0.05 μg/μg of DNA) for 20 minutes at 37°C. The single-stranded DNA fragments were phenol-ether extracted,ethanol precipitated, resuspended in 50 mM Tris buffer, pH 8,dephosphorylated with bacterial alkaline phosphatase and labeled with[γ⁻³² P]-ATP and polynucleotide kinase to a specific radioactivity of3×10⁶ cpm/μg DNA. After hybridization, the filters were washedsequentially with 2×SSC, and 1×SSC containing 1.0% SDS at 68° C. for 15minutes each, dried under a hot lamp, and counted in liquidscintillation fluid. To ensure that an excess of DNA probes was presentin the hybridization solution, several filters were prepared whichcontain different amounts of immobilized mRNA samples. The results ofthese hybridization experiments are summarized in FIGS. 7A and 7B. Thehalf-life of the penP mRNA produced from plasmid pSYC667 in either E.coli or B. subtilis was estimated to be about two minutes; that frompHCW-A3 was about six minutes. Analysis also revealed that the cellscarrying the plasmid pHCW-A3 have a higher steady state level of penPmRNA than the cell harboring the plasmid pSYC667. This result isexpected if the positive retroregulatory element stabilizes thecotranscribed gene. Since the increase in the levels of mRNA matches themagnitude of increase in penicillinse enzyme in both of these bacterialhosts as shown in Example III above, the positive retroregulatoryelement enhances gene expression through its influence on mRNAstability.

Deposited Strains

Deposits of strains listed in the following Table VI are stored in theMaster Culture Collection of Cetus Corporation, the assignee of thepresent application, Emeryville, Calif., U.S.A., and have been assignedthe Cetus Master Culture Collection numbers listed in the Table. Thelisted strains were also deposited by Cetus Corporation with theAmerican Type Culture Collection, Rockville, Md., U.S.A., on the datesindicated in the Table and were assigned the ATCC numbers listed in theTable. The ATCC deposits were made under the Budapest Treaty on theInternational Recognition of Deposits of Microorganisms for Purposes ofPatent Procedures and the Regulations promulgated thereunder, and thestrains will be available to the public in accordance with the terms ofsaid Treaty and Regulations.

                  TABLE VI                                                        ______________________________________                                                            ATCC                                                                          Deposit                                                   Strain    Plasmid   Number     Deposit Date                                   ______________________________________                                        E. coli   pSYC667   39758      July 3, 1984                                   K-12/CS412                                                                    B. thurin-          39756      July 3, 1984                                   gensis HD-1                                                                   E. coli   pHCW701   39757      July 3, 1984                                   K-12/MM294                                                                    E. coli   pFC54.t   39789      August 7, 1984                                 DG95 (λ)                                                               E. coli   pLW1      39405      August 4, 1983                                 MM294                                                                         E. coli             39626      March 6, 1984                                  MM294-1                                                                       E. coli             39452      September 26, 1983                             MM294                                                                         ______________________________________                                    

Various modifications of the invention, as described and exemplified inthe present specification, will be apparent to persons of skill in theart. It is intended that such modifications are within the scope of theinvention and the appended claims.

What is claimed is:
 1. A method for extending the half-life of a mRNAtranscript comprising:(a) providing a selected DNA sequence having apositive retroregulatory element ligated at the 3' end thereof, saidpositive retroregulator when transcribed into said mRNA transcripthaving an inverted repeat sequence capable of forming a stem and loopstructure having a ΔG° of about -30.4 Kcal/mole and wherein said elementfunctions in the 5' to 3' or 3' to 5' orientation to enhance expressionin prokaryotic cells; (b) providing a means for transcribing saidselected DNA sequence and said positive retroregulatory element ligatedthereto into said mRNA transcript wherein said means is selected fromthe group consisting of a prokaryotic cell and a prokaryotic cell freesystem; and (c) transcribing said selected DNA sequence having saidpositive retroregulatory element ligated at the 3' end thereof into saidmRNA transcript.
 2. The method of claim 1 wherein said mRNA transcriptcontains a translation termination signal sequence and said positiveretroregulatory element is in the 3' direction from said translationtermination signal sequence of said selected DNA sequence.
 3. The methodof claim 1 wherein said prokaryotic cell is Gram-positive.
 4. The methodof claim 3 wherein said Gram-positive cell is from the genus Bacillus.5. The method of claim 1 wherein said prokaryotic cell is Gram-negative.6. The method of claim 5 wherein said Gram-negative cell is an E. colicell.
 7. The method of claim 1 wherein said selected DNA sequence isheterologous to said host cell.
 8. The method of claim 1 wherein thepromotor controlling transcription of said selected DNA sequence isheterologous to said host cell.
 9. The method of claim 1 wherein thepromotor for controlling transcription of said selected DNA sequence ishomologous to a promotor native to said host cell.
 10. The method ofclaim 1 wherein the positive retroregulatory sequence is heterologous tosaid host cell.
 11. The method of claim 1 wherein said positiveretroregulatory sequence comprises a portion of the 3' flanking sequenceof the B. thuringiensis crystal protein gene and wherein said portioncomprises the sequence

    5'-AAAACGGACATCACCTCCATTGAAACGGAGTGATGTCCGTTTT-3'.


12. An mRNA sequence having an extended half-life conferred by aco-transcribed positive retroregulatory element, wherein said mRNAsequence is transcribed from a selected recombinant DNA sequence havinga positive retroregulatory element which comprises a portion of the 3'flanking sequence of the B. thuringiensis crystal protein gene andwherein said portion comprises the sequence5'-AAAACGGACATCACCTCCATTGAAACGGAGTGATGTCCGTTTT-3' and wherein saidportion is not native to said DNA sequence ligated at the 3' end thereofin the 5' or the 3' to 5' orientation and wherein said element whentranscribed is capable of forming a stem and loop structure having a ΔG°of about -30.4 Kcal/mole.